Phosphonic acid derivatives with metallopeptidase inhibitory activity

ABSTRACT

Compounds of formula (I) wherein R is an alkyl, aryl or arylalkyl group wherein the aryl is phenyl naphthyl or heterocycle; R 1  and R 2 , the same or different, are hydrogen atoms or alkyl groups; R 3  is an aryl or arylalkyl group wherein the aryl is as above; R 4  is a heterocycle optionally substituted with a heterocycle or phenyl, or it is a phenyl group substituted with a heterocycle (R 4  is not imidazole or indole); X is a bond or —OCONH or —CONH— group, processes for their preparation and pharmaceutical compositions thereof, are described. The compounds of formula (I) are endowed with a dual ACE-inhibitory and NEP-inhibitory activity and are useful in the treatment of cardiovascular disease.

The present invention relates to phosphonic acid derivatives useful inthe treatment of cardiovascular diseases and, more particularly, itrelates to phosphonic acid derivatives useful in the treatment ofcardiovascular diseases as metallopeptidase inhibitors. Thepharmacologic interest towards the study of metallopeptidase inhibitorymolecules derives from the role that said enzymes exert on the level ofthe cardiocirculatory system.

It is well-known in fact that compounds with angiotensin convertingenzyme (ACE) inhibitory activity are mainly useful in the treatment ofhypertension, of heart failure and of post-infarct in that they inhibitthe formation of angiotensin II, a substance which increases the bloodpressure.

Compounds with endothelin converting enzyme (ECE) inhibitory activityare useful as anti-vasoconstrictors in that they inhibit the formationof endothelin, a 21 amino acid peptide with vasoconstrictor activity.

Instead, compounds with inhibitory activity of the neutral endopeptidase(NEP) enzyme, also called enkephalinase, are useful as vasodilators anddiuretics in that the NEP enzyme is responsible for the inactivation,not only of endogenous enkephahne, but also of some natriuretic factorsamong which, for instance, the atrial natriuretic factor (ANF), avasodilating hormone secreted by heart.

Therefore, even exerting their action on the cardiovascular system withdifferent mechanisms of action, the compounds with metallopeptidaseinhibitory activity are generally used, alone or in combination, in thetreatment of hypertension, renal failure, congestive heart failure andpost-infarct.

In the U.S. Pat. No. 4,432,972 (E.R. Squibb & Sons, Inc.) phosphorylatedderivatives of amino acids such as, in particular, phosphonamidatesendowed with ACE-inhibitory and enkephahnase-inhibitory activity weredescribed.

Said compounds were described as useful hypotensive and analgesicagents. In the European patent application No. 0518299 (Takeda ChemicalIndustries, Ltd) some phosphonic acid derivatives, endowed withECE-inhibitory activity, usefull in the treatment of hypertension, ofcardiac or cerebrovascular diseases and of renal diseases weredescribed.

Now we have found phosphonic acid derivatives which are endowed withinhibitory activity on the angiotensin converting enzyme as well as onthe neutral endopeptidase enzyme (dual ACE/NEP-inhibitory activity)which renders them particularly useful in the cardiovascular therapy.

Therefore object of the present invention are the compounds of formula

wherein

R is a straight or branched C₁-C₆ alkyl group optionally substitutedwith one or more fluorine atoms, an aryl or arylalkyl group with from 1to 6 carbon atoms in the alkyl moiety wherein the aryl is a phenyl1-naphthyl, 2-naphthyl group or a 5 or 6 membered aromatic heterocyclewith 1 or 2 heteroatoms selected among nitrogen, oxygen and sulphur,optionally substituted with one or more substituents, the same ordifferent, selected among halogen atoms, hydroxy groups, alkyl, alkoxy,alkythio, alkylsulphonyl or alkoxycarbonyl groups with from 1 to 3carbon atoms in the alkyl moiety, carboxy groups, aminocarbonyl groups,acylamino groups, aminosulphonyl groups, mono- or di-alkylaminocarbonylgroups with from 1 to 3 carbon atoms in the alkyl moiety;

R₁ and R₂, the same or different, represent a hydrogen atom or astraight or branched C₁-C₄ alkyl group;

R₃ is a straight or branched C₁-C₆ alkyl group or an arylalkyl groupwith from 1 to 6 carbon atoms in the alkyl moiety wherein the aryl is aphenyl, 1-naphthyl 2-naphthyl group or a 5 or 6 membered aromaticheterocycle with one or two heteroatoms selected among nitrogen, oxygenand sulphur, optionally substituted as indicated for R;

R₄ is a 5 or 6 membered aromatic heterocyclic group with one or twoheteroatoms selected among nitrogen, oxygen and sulphur, optionallysubstituted with a 5 or 6 membered aromatic heterocyclic group with oneor two heteroatoms selected among nitrogen, oxygen and sulphur or with aphenyl group, or it is a phenyl group substituted with a 5 or 6 memberedaromatic heterocyclic group with one or two heteroatoms selected amongnitrogen, oxygen and sulphur, being the phenyl and the heterocyclicgroups optionally substituted with one or more substituents, the same ordifferent, selected among halogen atoms, alkyl alkoxy, alkylthio oralkoxycarbonyl groups with from 1 to 3 carbon atoms in the alkyl moiety;

X is a single bond or an —O—CONH— or —CONH— group;

the carbon atoms marked with an asterisk are asymmetric carbon atoms;

and pharmaceutically acceptable salts thereof;

provided that R₄ is not an imidazolyl or indolyl group.

The compounds of formula I contain at least two asymmetric carbon atomsand can thus exist in the form of stereoisomers.

Therefore, object of the present invention are the compounds of formulaI in the form of stereoisomeric mixture as well as in the form of singlestereoisomers.

The compounds of formula I object of the present invention are endowedwith a dual ACE/NEP-inhibitory activity and are useful in the treatmentof cardiovascular diseases.

In the present description, unless otherwise specified, with the termalkyl group we intend a straight or branched alkyl such as methyl,ethyl, n.propyl, isopropyl, n.butyl, sec-butyl tert-butyl isobutyln.pentyl 2-pentyl, 3-pentyl, isopentyl, tert-pentyl n.hexyl andisohexyl; with the term alkoxy group we intend a straight or branchedalkoxy such as methoxy, ethoxy, n.propoxy and isopropoxy; with the termhalogen atom we intend a fluorine, chlorine, bromine or iodine atom;with the term acyl we intend an acyl group deriving from an aliphatic oraromatic carboxylic acid such as acetic, propionic, butyric and benzoicacid; with the term aryl we intend an aromatic group such as phenyl,1-naphthyl, 2-naphthyl or a 5 or 6 membered heterocyclic groupcontaining 1 or 2 heteroatoms selected among nitrogen, oxygen andsulphur such as thiazole, isoxazole, oxazole, isothiazole, pyrazole,imidazole, thiophene, pyrrole, pyridine, pyrimidine, pyrazine and furan,optionally benzocondensed.

Examples of pharmaceutically acceptable salts of the compounds offormula I are the salts with alkali or alkali-earth metals and the saltswith pharmaceutically acceptable organic bases.

Preferred compounds of formula I are the compounds wherein R₄ representsa phenyl group substituted in position 4 with a heterocyclic group.

Particularly preferred, in this class, are the compounds of formula Iwherein R₁ and R₂ represent a hydrogen atom and R₃ represents a straightor branched C₁-C₄ alkyl group.

Preferred examples of pharmaceutically acceptable salts of the compoundsof formula I are the salts with alkali metals such as sodium, lithiumand potassium.

Specific examples of preferred compounds of formula I, object of thepresent invention, are:

N-(N′-propylphosphonyl-leucyl)-[4-(2-furyl)]-phenylalanine;

N-(N′-propylphosphonyl-leucyl)-[4-(3-furyl)]-phenylalanine;

N-(N′-propylphosphonyl-leucyl)-[4-(2-thienyl)]-phenylalanine;

N-(N′-propylphosphonyl-leucyl)-[4-(3-thienyl)]-phenylalanine;

N-(N′-propylphosphonyl-leucyl)-[4-(N″-methyl-2-pyrrolyl)]-phenylalanine;

N-(N′-propylphosphonyl-leucyl)-[4-(N″-methyl-3-pyrrolyl)]-phenylalanne;

N-(N′-propylphosphonyl-leucyl)-[4-(2-thiazolyl)]-phenylalanine;

N-(N′-propylphosphonyl-leucyl)-[4-(2-pyridyl)]-phenylalanine;

N-(N′-propylphosphonyl-leucyl)-[4-(3-pyridyl)]-phenylalanine;

N-(N′-propylphosphonyl-leucyl)-(4-pyrazinyl)-phenylalanine;

N-(N′-propylphosphonyl-leucyl)-[4-(5-pyrmidinyl)]-phenylalanine;

N-(N′-propylphosphonyl-valyl)-[4-(2-furyl)]-phenylalaine;

N-(N′-propyhphosphonyl-valyl)-[4-(3-furyl)]-phenylalanine;

N-(N′-propyhphosphonyl-valyl)-[4-(2-thienyl)]-phenylalanine;

N-(N′-propylphosphonyl-valyl)-[4-(3-thienyl)]-phenylalanine;

N-(N′-propylphosphonyl-valyl)-[4-(N″-methyl-2-pyrrolyl)]-phenylalanine;

N-(N′-propylphosphonyl-valyl)-[4-(N″-methyl-3-pyrrolyl)]-phenylalanine;

N-(N′-propylphosphonyl-valyl)-[4-(2-thiazolyl)]-phenylalanine;

N-(N′-propylphosphonyl-valyl)-[4-(2-pyridyl)]-phenylalanine;

N-(N′-propylphosphonyl-valyl)-[4-(3-pyridyl)]-phenylalanine;

N-(N′-propylphosphonyl-valyl)(4-pyrazinyl)-phenylalanine;

N-(N′-propylphosphonyl-valyl)-[4-(5-pyrimidinyl)]-phenylalanine;

N-[N′-(2-thienylmethyl)phosphonyl-leucyl]-[4-(2-furyl)]-phenylalanine;

N-[N′-(2-thienylmethyl)phosphonyl-leucyl]-[4-(3-fiuyl)]-phenylalanine;

N-[N′-(2-thienylmethyl)phosphonyl-leucyl]-[4-(2-thienyl)]-phenylalanine;

N-[N′-(2-thienylmethyl)phosphonyl-leucyl]-[4-(3-thienyl)]-phenylalanine;

N-[N′-(2-thienylmethyl)phosphonyl-leucyl]-[4-(N″-methyl-2-pyrrolyl)]-phenylalanine;

N-[N′-(2-thienylmethyl)phosphonyl-leucyl]-[4-(N″-methyl-3-pyrrolyl)]-phenylalanine;

N-[N′-(2-thienylmethyl)phosphonyl-leucyl]-[4-(2-thiazolyl)]-phenylalanine;

N-[N′-(2-thienylmethyl)phosphonyl-leucyl]-[4-(2-pyridyl)]-phenylalanine;

N-[N′-(2-thienylmethyl)phosphonyl-leucyl]-[4-(3-pyridyl)]-phenylalanine;

N-[N′-(2-thienylmethyl)phosphonyl-leucyl]-(4-pyrazinyl)-phenylalanine;

N-[N′-(2-thienylmethyl)phosphonyl-leucyl]-[4-(5-pyrimidinyl)]-phenylalanine;

N-[N′-(2-thienylmethyl)phosphonyl-valyl]-[4-(2-furyl)]-phenylalanine;

N-[N′-(2-thienylmethyl)phosphonyl-valyl]-[4-(3-furyl)]-phenylalanine;

N-[N′-(2-thienyhnethyl)phosphonyl-valyl]-[4-(2-thienyl)]-phenylalanine;

N-[N′-(2-thienylmethyl)phosphonyl-valyl]-[4-(3-thienyl)]-phenylalanine;

N-[N′-(2-thienylmethyl)phosphonyl-valyl]-[4-(N″-methyl-2-pyrrolyl)]-phenylalanine;

N-[N′-(2-thienylmethyl)phosphonyl-valyl]-[4-(N″-methyl-3-pyrrolyl)]-phenylalanine;

N-[N′-(2-thienylmethyl)phosphonyl-valyl]-[4-(2-thiazolyl)]-phenylalanine;

N-[N′-(2-thienylmethyl)phosphonyl-valyl]-[4-(2-pyridyl)]-phenylalanine;

N-[N′-(2-thienylmethyl)phosphonyl-valyl]-[4-(3-pyridyl)]-phenylalanine;

N-[N′-(2-thienyhmethyl)phosphonyl-valyl]-(4-pyrazinyl)-phenylalanine;

N-[N′-(2-thienylmethyl)phosphonyl-valyl]-[4-(5-pyrmidinyl)]-phenylalanine;

N-[N′-(benzyloxycarbonylaminomethyl)phosphonyl-leucyl]-[4-(2-furyl)]-phenylalanine;

N-[N′-(benzyloxycarbonylaminomethyl)phosphonyl-leucyl]-[4-(3-furyl)]-phenylalanine;

N-[N′-(benzyloxycarbonylaminomethyl)phosphonyl-leucyl]-[4-(2-thienyl)]-phenylalanine;

N-[N′-(benzyloxycarbonylaminomethyl)phosphonyl-leucyl]-[4-(3-thienyl)]-phenylalanine;

N-[N′-(benzyloxycarbonylaminomethyl)phosphonyl-leucyl)-[4-(N″methyl-2-pyrrolyl)]-phenylalanine;

N-[N′-(benzyloxycarbonylaminomethyl)phosphonyl-leucyl]-[4-(N″-methyl-3-pyrrolyl)]-phenylalanine;

N-[N′-(benzyloxycarbonylaminomethyl)phosphonyl-leucyl]-[4-(2-thiazolyl)]-phenylalanine;

N-[N′-(benzyloxycarbonylaminomethyl)phosphonyl-leucyl]-[4-(2-pyridyl)]-phenylalanine;

N-[N′-(benzyloxycarbonylaminomethyl)phosphonyl-leucyl]-[4-(3-pyridyl)]-phenylalanine;

N-[N′-(benzyloxycarbonylaminomethyl)phosphonyl-leucyl]-(4-pyrazinyl)-phenylalanine;

N-[N′-(benzyloxycarbonylaminomethyl)phosphonyl-leucyl]-[4-(5-pyrimidinyl)]-phenylalanine;

N-[N′-(benzyloxycarbonylaminomethyl)phosphonyl-valyl]-[4-(2-furyl)]-phenylalanine;

N-[N′-(benzyloxycarbonylaminomethyl)phosphonyl-valyl]-[4-(3-furyl)]-phenylalanine;

N-[N′-(benzyloxycarbonylaminomethyl)phosphonyl-valyl]-[4-(2-thienyl)]-phenylalanine;

N-[N′-(benzyloxycarbonylaminomethyl)phosphonyl-valyl]-[4-(3-thienyl)]-phenylalanine;

N-[N′-(benzyloxycarbonylaminomethyl)phosphonyl-valyl]-[4-(N″-methyl-2-pyrrolyl)]-phenylalanine;

N-[N′-(benzyloxycarbonylaminomethyl)phosphonyl-valyl]-[4-(N″-methyl-3-pyrrolyl)]-phenylalanine;

N-[N′-(benzyloxycarbonylaminomethyl)phosphonyl-valyl]-[4-(2-thiazolyl)]-phenylalanine;

N-[N′-(benzyloxycarbonylaminomethyl)phosphonyl-valyl]-[4-(2-pyridyl)]-phenylalanine;

N-[N′-(benzyloxycarbonylaminomethyl)phosphonyl-valyl]-[4-(3-pyridyl)]-phenylalanine;

N-[N′-(benzyloxycarbonylaminomethyl)phosphonyl-valyl]-(4-pyrazinyl)-phenylalanine;

N-[N′-(benzyloxycarbonylaminomethyl)phosphonyl-valyl]-[4-(5-pyrimidinyl)]-phenylalanine;

N-[N′-(acetylaminomethyl)phosphonyl-leucyl]-[4-(2-furyl)]-phenylalanine;

N-[N′-(acetylaminomethyl)phosphonyl-leucyl]-[4-(3-furyl)]-phenylalanine;

N-[N′-(acetylaminomethyl)phosphonyl-leucyl]-[4-(2-thienyl)]-phenylalanine;

N-[N′-(acetylaminomethyl)phosphonyl-leucyl]-[4-(3-thienyl)]-phenylalanine;

N-[N′-(acetylaminomethyl)phosphonyl-leucyl]-[4-(N″-methyl-2-pyrrolyl)]-phenylalanine;

N-[N′-(acetylaminomethyl)phosphonyl-leucyl]-[4-(N″-methyl-3-pyrrolyl)]-phenylalanine;

N-[N′-(acetylaminomethyl)phosphonyl-leucyl]-[4-(2-thiazolyl)]-phenylalanine;

N-[N′-(acetylaminomethyl)phosphonyl-leucyl]-[4-(2-pyridyl)]-phenylalanine;

N-[N′-(acetylaminomethyl)phosphonyl-leucyl]-[4-(3-pyridyl)]-phenylalanine;

N-[N′-(acetylaminomethyl)phosphonyl-leucyl]-(4-pyrazinyl)-phenylalanine;

N-[N′-(acetylaminomethyl)phosphonyl-leucyl]-[4-(5-pyrimidinyl)]-phenylalanine;

N-[N′-(acetylaminomethyl)phosphonyl-valyl]-[4-(2-furyl)]-phenylalanine;

N-[N′-(acetylaminomethyl)phosphonyl-valyl]-[4-(3-furyl)]-phenylalanine;

N-[N′-(acetylaminomethyl)phosphonyl-valyl]-[4-(2-thienyl)]-phenylalanine;

N-[N′-(acetylaminomethyl)phosphonyl-valyl]-[4-(3-thienyl)]-phenylalanine;

N-[N′-(acetylaminomethyl)phosphonyl-valyl]-[4-(N″-methyl-2-pyrrolyl)]-phenylalanine;

N-[N′-(acetylaminomethyl)phosphonyl-valyl]-[4-(N″-methyl-3-pyrrolyl)]-phenylalanine;

N-[N′-(acetylaminomethyl)phosphonyl-valyl]-[4-(2-thiazolyl)]-phenylalanine;

N-[N′-(acetylaminomethyl)phosphonyl-valyl]-[4-(2-pyridyl)]-phenylalanine;

N-[N′-(acetylaminomethyl)phosphonyl-valyl]-[4-(3-pyridyl)]-phenylalanine;

N-[N′-(acetylaminomethyl)phosphonyl-valyl]-(4-pyrazinyl)-phenylalanine;

N-[N′-(acetylaminomethyl)phosphonyl-valyl]-[4-(5-pyrimidinyl)]-phenylalanine.

The preparation of the compounds of formula I, object of the presentinvention, comprises the reaction between a phosphorylated derivative offormula

wherein

R, R₁ and X have the above reported meanings, W represents a halogenatom, preferably chlorine, and Y represents a protective group,preferably a C₁-C₄ alkyl, a phenyl or a phenylalkyl with from 1 to 4carbon atoms in the alkyl moiety;

and a dipeptide derivative of formula

 wherein

R₂, R₃ and R₄ have the above reported meanings.

The phosphorylated derivative of formula II can be prepared from thecorresponding compound of formula

wherein

R, R₁ and X have the above reported meanings and the Z groups bothrepresent a W or OY group wherein W and Y have the above reportedmeanings.

The preparation of the compounds of formula II from the correspondingcompounds of formula IV is carried out according to conventionaltechniques, by reaction with a halogenating agent or with a compound offormula YOH, wherein Y has the above reported meanings.

For a reference to the preparation of the compounds of formula IIwherein X is a single bond or an —O—CONH— group see, for instance, D. S.Karanewsky et at, J. Med. Chem. 1988, 31, 204-212 and B. P. Morgan etal, J. Am Chem. Soc. 1991, 113, 297-307.

The compounds of formula II wherein X is a —CONH— group can be prepared,as example, from the corresponding compounds of formula II wherein X isan —O—CONH— group and R=benzyl through hydrogenolysis of the carbamicgroup (R—O—CONH—) and subsequent reaction with a compound of formula

R—COW₁  (V)

wherein

R has the above reported meanings and W₁ represents a chlorine orbromine atom.

The dipeptide derivatives of formula III, in their turn, can be preparedthrough the condensation between an amino acid of formula

wherein

R₂ and R₃ have the above reported meanings;

and an alanine derivative of formula

 wherein R₄ has the above reported meanings.

The condensation reaction is carried out according to conventionaltechniques of the chemistry of peptides.

Before carrying out the reaction it can be useful to properly protectthe optional functional groups which could interfere in the reaction.

The optional protection is carried out according to conventionaltechniques.

For instance, in the reaction between the phosphorylated derivative offormula II and the dipeptide derivative of formula III it can be usefulto protect the free carboxy function of the compound of formula III aswell as the hydroxy function of the phosphonic group.

Likewise, in the reaction between the amino acid of formula VI and thealanine derivative of formula VII, it can be useful to protect the aminofunction of the derivative of formula VI and the carboxy function of thederivative of formula VII.

The evaluation of the usefulness of the optional protection as well asthe selection of the kind of adopted protection according to thereaction to be carried out and to the functional groups to be protectedare within the normal knowledge of the man skilled in the art.

The removal of the optional protective groups is carried out accordingto conventional techniques.

For a general reference to the use of protective groups in organicchemistry see Theodora W. Greene and Peter G. M. Wuts “Protective Groupsin Organic Synthesis”, John Wiley & Sons, Inc., II Ed., 1991.

The compounds of formula VI and VII are known or easily preparedaccording to known methods.

For a reference to the preparation of the compounds of formula VII see,for instance, the synthetic methods described by W. C. Shieh and T. R.Bailey in J. Org. Chem. 1992, 57, 379-381 and Tetrahedron Letters, 27,4407-4410, 1986, respectively.

The compounds of formula I, object of the present invention, can befurther prepared according to an alternative synthetic scheme comprisingthe reaction between a phosphorylated derivative of formula

wherein

R, R₁, R₂, R₃, X and Y have the above reported meanings;

and an alanine derivative of formula VII.

Analogously to what previously reported, also in the above reaction itcan be useful to protect, according to conventional techniques, eventualfunctional groups which could interfere in the reaction.

The compounds of formula VIII are known or easily prepared according toknown methods.

For instance, the compounds of formula VIII can be prepared through thereaction between the phosphorylated derivative of formula II and theamino acid of formula VI according to conventional methods of thechemistry of peptides.

In view of what above indicated, it is clear to the man skilled in theart that the preparation of the compounds of formula I wherein X is a—CONH— group can be optionally carried out starting from thecorresponding compounds of formula I wherein X is an —O—CONH— group andR=benzyl, prepared according to one of the aforementioned syntheticmethods.

The compounds of formula I in the form of single stereoisomers areprepared by stereo selective synthesis or by separation of thestereoisomeric mixture according to conventional techniques.

Also the preparation of the salts of the compounds of formula I, objectof the invention, is carried out according to conventional techniques.

The compounds of formula I object of the present invention are endowedwith a dual ACE/NEP-inhibitory activity and are useful in the treatmentof cardiovascular diseases.

The inhibitory activity of the compounds of formula I, in particular,was evaluated by means of in vitro and ex vivo tests.

The in vitro inhibitory activity of the compounds of formula I wasevaluated in comparison to known molecules endowed with ACE-inhibitoryor NEP-inhibitory activity (example 3).

Captopril, a drug known as the first orally active ACE-inhibitor (TheMerck Index, XI ed.—No. 1773, pages 267-268), was used as a comparisoncompound for the ACE-inhibitory activity.

Thiorphan [DL-(3-mercapto-2-benzylpropionyl)glycine] instead, knownmolecule considered the parent compound for NEP-inhibitors and describedfor the first time by Roques et al. in Nature, vol. 288, pages 286-288,(1980), was used as a comparison compound for the NEP-inhibitoryactivity.

N-[N′-(4-phenyl)butylphosphonyl-L-phenylalanyl]-L-phenylalaninedilithium salt (hereinafter referred to as compound R-1), exemplified inthe aforementioned U.S. Pat. No. 4,432,972, was considered as a furthercomparison compound for evaluating the in vitro inhibitory activity ofthe compounds of formula I.

The in vitro inhibitory activity of the compounds of formula I,expressed as IC₅₀ value, is pharmacologically significant in that itresults at nM concentrations.

Said activity resulted to be at least comparable to that of Captopril,to what it concerns the ACE-inhibitory activity, and to that ofThiorphan, to what it concerns the NEP-inhibitory activity.

With respect to the compound R-1, moreover, the dual ACE/NEP-inhibitoryactivity of the compounds of formula I resulted to be significantlyhigher.

As above indicated, the inhibitory activity of the compounds of formulaI was evaluated also by means of ex vivo experiments, using theaforementioned compound R-1 as a comparison compound (example 4).

The dual ACE/NEP-inhibitory activity ex vivo of the compounds of formulaI resulted to be significantly higher than that of the comparisoncompound.

For a practical use in therapy, the compounds of formula I can beformulated in solid or liquid pharmaceutical compositions, suitable tooral or parenteral administration.

Therefore, the pharmaceutical compositions containing a therapeuticallyeffective amount of a compound of formula I in admixture with a carrierfor pharmaceutical use are a further object of the present invention.

Specific examples of pharmaceutical compositions according to thepresent invention are tablets, coated tablets, capsules, granulates,solutions and suspensions suitable to oral administration, solutions andsuspensions suitable to parenteral administration.

The pharmaceutical compositions object of the present invention areprepared according to conventional techniques.

Although the compounds of formula I are active as such, with the aim tosatisfy particular therapeutic or pharmaceutical properties, it can beuseful to transform them into the corresponding biologic precursors(pro-drugs).

Therefore, according to the conventional techniques for the preparationof pro-drugs of phosphorylated and amido derivatives, suitable pro-drugscan be obtained for instance through the esterification of the carboxyfunction or of the phosphonic function.

Also the compounds of formula I in the form of pro-drugs and, inparticular, the compounds obtained through the esterification of thecarboxy or phosphonic function, as well as the pharmaceuticalcompositions which contain the compounds of formula I in the form ofpro-drugs and, in particular, which contain the compounds of formula Iwherein the carboxy or phosphonic group results to be esterified, arewithin the scope of the present invention.

The daily dose of the compound of formula I or of the correspondingpro-drug will depend on several factors such as the seriousness of thedisease, the individual response of the patient or the kind offormulation but it is usually comprised between 0.01 mg and 20 mg per kgof body weight divided into a single dose or into more daily doses.

With the aim of illustrating the present invention the followingexamples are now given.

Unless otherwise specified, the flash chromatographies were carried outby using flash chromatography silica gel from Baker company (code7024-00).

REFERENCE EXAMPLE 1N-[N′-(4-phenyl)butylphosphonyl-L-phenlalanyl]-L-phenylalanine dilithiumsalt (Compound R-1)

Said compound was prepared according to the procedure described in U.S.Pat. No. 4,432,972 (example 36).

EXAMPLE 1 N-(N′-propylphosphonyl-L-leucyl)-4-(2-furyl)]-L-phenylalaninedilithium salt (Compound 1)

a) Preparation ofN-[N′-(tert-butoxycarbonyl)-L-leucyl]-[4-(2-furyl)]-L-phenylalaninemethyl ester

Dicyclohexylcarbodiimide (1.75 g; 8.50 mmoles) was added to a solutionof N-(tert-butoxycarbonyl)-L-leucine monohydrate (0.98 g; 3.93 mmoles)and N-hydroxysuccinimide (0.45 g; 3.93 mmoles) in 1,4-dioxane (40 ml).

The reaction mixture was kept under stirring for a hour at roomtemperature and, subsequently, the formed dicyclohexylurea was filteredoff.

(2-furyl)]-L-phenylalanine methyl ester (0.75 g; 3.27 mmoles), preparedaccording to the synthetic method described by W. C. Shieh in J. Org.Chem, 1992, 57, 379-381, was then added to the obtained solution.

The reaction mixture was kept under stirring for 18 hours and,subsequently, the solvent was evaporated at reduced pressure.

The obtained residue was collected with ethyl ether and the mixture wasfiltered again.

The solvent was thus evaporated at reduced pressure and the resultantcrude was purified by silica gel flash chromatography (petroleumether:ethyl acetate=75:25; pressure of nitrogen 0.1 atm) furnishingN-[N′-(tert-butoxycarbonyl)-L-leucyl]-[4-(2-furyl)]-L-phenylalaninemethyl ester (0.98 g; 30% yield), as a white solid.

¹H-NMR (200 MHz, CDCl₃): δ (ppm): 0.85-0.93 (2d, 6H); 1.4 (s, 9H);1.50-1.70 (m, 3H); 3.00-3.20 (m, 2H); 3.70 (m, 3H); 4.00-4.12 (m, 1H);4.70-4.90 (m, 2H); 6.52 (d, 1H); 6.41-7.41 (m, 3H); 7.1-7.6 (m, 4H).

b) Preparation of N-(L-leucyl)-[4-(2-furyl)]-L-phenylalanine methylester hydrochloride

Thionyl chloride (0.31 ml; 4.28 mmoles) was added at 0° C. to a solutionof N-[N′-(tert-butoxycarbonyl)-L-leucyl]-[4-(2-furyl)]-L-phenylalaninemethyl ester (0.98 g; 2.14 mmoles), prepared as described in example 1a,in methanol (20 ml).

The reaction mixture was kept under stirring at room temperature for 24hours and the solvent was then evaporated at reduced pressure furnishingN-(L-leucyl)-[4-(2-furyl)]-L-phenylalanine methyl ester hydrochloride(0.79 g; 93% yield), used as such in the subsequent reaction.

c) Preparation ofN-[N′-(phenoxy)(propyl)phosphoryl-L-leucyl]-[4-(2-furyl)]-L-phenylalanine

A solution of phenol (0.28 g; 3.00 mmoles) and triethylamine (0.42 ml;3.00 mmoles) in methylene chloride (17 ml) was slowly added dropwise toa solution of propylphosphonic dichloride (0.37 ml; 3.00 mmoles) inmethylene chloride (4 ml), cooled at 0° C. and under nitrogen.

A mixture of N-(L-leucyl)-[4-(2-furyl)]-L-phenylalanine methyl esterhydrochloride (0.79 g; 2.00 mmoles), prepared as described in example1b, and triethylamine (0.7 ml; 5.00 mmoles) in methylene chloride (5 ml)was then added dropwise to the reaction mixture, kept under stirring atroom temperature for 3 hours and subsequently cooled at 0° C.

The reaction mixture was kept under stirring at room temperature for 3hours and water was then added.

The phases were separated and the organic phase was dried on sodiumsulphate and evaporated at reduced pressure.

The resultant residue was purified by silica gel flash chromatography(eluent petroleum ether:ethylacetate=1:1; pressure of nitrogen 0.1 atm)finishingN-[N′-(phenoxy)(propyl)phosphoryl-L-leucyl]-[4-(2-furyl)]-L-phenylalaninemethyl ester (0.4 g; 40% yield), as a white solid.

¹H-NMR (200 MHz, CDCl₃): δ (ppm): 0.70-1.90 (m, 16H); 2.90-3.20 (m, 2H);3.35-3.51 (m, 1H); 3.65 (s, 3H); 3.70-3.90 (m, 1H); 4.65-4.80 (m, 1H);6.70-6.85 (m, 1H); 6.40-7.40 (m, 3H); 7.00-7.60 (m, 9H).

d) Preparation ofN-(N′-propylphosphonyl-L-leucyl)-[4(2-furyl)]-L-phenpylalanine dilithiumsalt

A solution of lithium hydroxide monohydrate (90 mg; 2.16 mmoles) inwater (2 ml) was added to the solution ofN-[N′-(phenoxy)(propyl)phosphoryl-L-leucyl]-[4-(2-furyl)]-L-phenylalaninemethyl ester (0.4 g; 0.72 mmoles), prepared as described in example 1c,in tetrahydrofuran (5 ml).

The reaction mixture was kept under stirring at room temperature for ahour.

The mixture was subsequently evaporated at reduced pressure and theobtained residue was collected with ethanol and evaporated again,repeating twice this procedure.

The residue was then collected with ethyl acetate and the mixture waskept under stirring at room temperature for 24 hours.

The mixture was then filtered affordingN-(N′-propylphosphonyl-L-leucyl)-[4-(2-furyl)]-L-phenylalanine dilithiumsalt (0.3 g; 90% yield) as a white solid.

¹H-NMR (200 MHz, D₂O): δ (ppm): 0.58-0.73 (m, 9H); 0.98-1.46 (m, 7H);2.71-3.11 (m, 2H); 3.26-3.38 (m, 1H); 4.27-4.33 (m, 1H); 6.39 (dd, 1H);6.63 (d, 1H); 7.33 (d, 1H); 7.10-7.52 (m, 4H).

EXAMPLE 2N-(N′-propylphosphonyl-L-valyl)-[4-(2-thiazolyl)]-L-phenylalaninedilithium salt (Compound 2)

By working as described in example 1 through steps a-d, the followingcompounds were prepared:

a) Preparation ofN-[N-′-(tert-butoxycarbonyl)-L-valyl]-[4-(2-thiazolyl)]-L-phenylalaninemethyl ester

¹H-NMR (200 MHz, CDCl₃): δ (ppm): 0.80-0.95 (m, 6H, CH ₃—CH—CH ₃); 1.42[s, 9H, C(CH₃)₃]; 2.00-2.19 (m, 1H, CH₃—CH—CH₃); 3.03-3.22 (m, 2H, CH₂-phenylene); 3.70 (s, 3H, COOCH₃); 3.83-3.93 (m, 1H, NH—CH—CH);4.72-5.05 (m, 2H, NHCOO, CHCOO); 6.40 (bd, 1H, NH—CH—COO); 7.13-7.90 (m,6H, aryl).

b) Preparation of N-(L-valyl)-[4-(2-thiazolyl)]-L-phenylalanine methylester hydrochloride

¹H-NMR (200 MHz, D₂O): δ (ppm): 0.77-0.83 (m, 6H, CH ₃—CH—CH ₃);1.93-2.10 (m, 1H, CH₃—CH—CH₃); 2.96-3.21 (m, 2H, CH ₂-phenylene); 3.55(s, 3H, COOCH₃); 3.62 (d, 1H, CH-NH₂); 4.60-4.70 (m, 1H, CH—COO);7.25-7.74 (m, 4H, phenylene); 7.64-7.87 (m, 2H, thiazolyl).

c) Preparation ofN-[N′-(phenoxy)(propyl)phosphoryl-L-valyl]-[-4-(2-thiazolyl)]-L-phenylalaninemethyl ester

¹H-NMR (200 MHz, CDCl₃): δ (ppm): 0.70-1.07 (m, 9H, CH ₃—CH—CH ₃, CH₃—CH₂); 1.50-2.00 (m, 5H, CH ₂—CH ₂—P—NH—CH—CH); 2.90-3.18 (m, 2H, CH₂-phenylene); 3.22-3.70 (m, 2H, P—NH—CH); 3.64 (s, 3H, COOCH₃);4.71-4.87 (m, 1H, CH—COO); 6.75-6.86 (m, 1H, NHCO); 7.05-7.89 (m, 11H,aryl).

d) Preparation ofN-(N′-propylphosphonyl-L-valyl)-[4-(2-thiazolyl)]-L-phenylalaninedilithium salt

¹H-NMR (200 MHz, D₂O): δ (ppm): 0.40-0.80 (m, 9H, CH ₃—CH—CH ₃, CH₃—CH₂); 1.00-1.70 (m, 5H, CH ₂—CH ₂—P—NH—CH—CH); 2.75-3.24 (m, 3H,P—NH—CH, CH ₂-phenylene); 4.25-4.39 (m, 1H, CH—COO); 7.16-7.70 (m, 6H,aryl).

EXAMPLE 3 In vitro Evaluation of the Pharmacologic Activity

a) NEP-inhibitory activity

The NEP-inhibitory activity was evaluated in rat kidney cortex membranesprepared according to the procedure described by T. Maeda et al. inBiochim Biophys. Acta 1983, 731(1), 115-120.

By working at 0-4° C. kidneys were removed from male Sprague-Dawley ratsweighing approximately 300 g.

Cortex was carefully dissected, finely minced and suspended in ahomogenization buffer (10 mM sodium phosphate pH 7.4 containing 1 mMMgCl2, 30 mM NaCl, 0.02% NaN3) 1:15 weight/volume.

The tissue was then homogenized for 30 seconds using an Ultra-Turraxhomogenizer.

Approximately 10 ml of homogenate were layered over 10 ml of sucrose(41% weight/volume) and centrifuged at 31200 rpm for 30 minutes at 4° C.in a fixed angle rotor.

The membranes were collected from the buffer/sucrose interface, washedtwice with 50 mM TRIS/HCl buffer (pH 7.4) and resuspended into the samebuffer for storage.

The membranes were stored in small aliquots at −80° C. until use.

The NEP-inhibitory activity was evaluated according to the methoddescribed by C. Llorens et al., in Eur. J. Pharmacol., 69, (1981),113-116, as reported hereinafter.

Aliquots of the membrane suspension prepared as above described(concentration 5 μg/ml of proteins) were preincubated in the presence ofan aminopeptidase inhibitor (Bestatin−1 mM) for 10 minutes at 30° C.[³H][Leu⁵]-enkephaline (15 nM) and buffer TRIS/HCl pH 7.4 (50 mM) wereadded in order to obtain a final volume of 100 μl.

Incubation (20 minutes at 30° C.) was stopped by adding HCl 0.1M (100μl).

The formation of the metabolite [³H]Tyr-Gly-Gly was quantified, afterseparation of the unreacted substrate by chromatography on polystirenecolumns (Porapak Q), by measuring the relative radioactivity throughliquid scintillation.

The percentage of inhibition of the metabolite formation in the membranepreparations treated with the compounds of formula I and with thecomparative compounds with respect to the untreated membranepreparations was expressed as IC₅₀ (nM) value.

b) ACE-inhibitory activity

The ACE-inhibitory activity was evaluated according to the methodreported in the literature by B. Holmquist et al., in AnalyticalBiochemistry 95, 540-548 (1979).

50 μM of ACE (250 mU/ml purified by lung rabbit, EC 3.4.15.1 SIGMA) werepreincubated with 50 μl of the compounds of formula I or with thecomparison compounds in thermostated cuvettes at 37° C.

The reaction was started by addingfurylacryloylphenylalanylglycylglycine 0.8 mM (FAPGG-SIGMA).

Contemporaneously, by using a Beckman DU-50 spectrophotometer providedwith a program for calculating delta A/minutes and regressioncoefficients of the enzyme kinetics curves, the absorbance at 340 nm wasrecorded in continuo for 5 minutes.

The inhibition of the enzymatic activity of the compounds of formula Iand of the comparison compounds was expressed as IC₅₀ (nM) value.

The compounds of formula I were tested as lithium salts.

The IC₅₀ (nM) values related to the ACE-inhibitory and NEP-inhibitoryactivity of the compounds 1 and 2 and of the comparison compounds R-1,Thiorphan and Captopril are reported in the following table 1.

Table 1

NEP-inhibitory and ACE-inhibitory activity, expressed as IC₅₀ (nM)value, of the compound 1, of the compound 2, of the compound R-1, ofThiorphan and of Captopri.

ACE-inhibitory activity NEP-inhibitory activity Compound IC₅₀ (nM) IC₅₀(nM) 1 5.7 6.0 2 9.4 2.7 R-1 20.0 5.5 Thiorphan 98.6 11.3  Captopril 2.8not active

The data reported in table 1 show that the compounds of formula I,object of the present invention, are endowed with a significant dualACE/NEP-inhibitory activity. Said activity resulted to be comparable tothat of Captopril, to what it concerns the ACE-inhibitory activity, andto that of Thiorphan, to what it concerns the NEP-inhibitory activity.

Moreover, the dual ACE/NEP-inhibitory activity of the compounds offormula I resulted to be significantly higher than that of the compoundR-1.

EXAMPLE 4 Ex vivo Evaluation of the Pharmacologic Activity

a) NEP-inhibitory activity

The ex vivo NEP-inhibitory activity was evaluated according to themethod reported in the literature by M. Orlowsky et aL, in Biochemistry1981, 20, 4942-4950.

The inhibitory activity of the compounds of formula I and of thecompound R-1 was evaluated in kidneys of spontaneously hypertensive rats(SHR), 5 minutes after i.v. injection of the tested compounds (21μmoles/Kg).

After the removal of the kidneys from SHR, the renal tissue washomogenized and incubated for 15 minutes at 37° C. in the presence ofGlutaryl-Ala-Ala-Phe-2-naphthyliamide (GAAP), as a substrate, andaminopeptidase M at pH 7.6. The reaction was stopped by adding anaqueous solution at 10% of trichloroacetic acid.

The released 2-naphthylamine was determined by adding fast garnet dye (2ml). Enzyme reaction rates were determined by measuring the increase inthe optical density at 524 nm (OD₅₂₄) with respect to a standardobtained with 2-naphthylamine complexed with fast garnet.

The NEP-inhibitory activity of the compounds of formula I and of thecompound R-1 was expressed as percentage of inhibition in SHR kidneys.

b) ACE-inhibitory activity

The ex vivo ACE-inhibitory activity was evaluated by using a radiometricmethod, as reported in the literature by J. W. Ryan et al. in Biochem J.(1977), 167, 501-504.

The inhibitory activity of the compounds of formula I and of thecompound R-1 was evaluated in lungs of spontaneously hypertensive rats(SHR), 5 minutes after i.v. injection of the tested compounds (21μmoles/Kg).

After the removal of the lungs from SHR, the lung tissue was homogenizedand incubated for 2 hours at 37° C. in the presence of [³H]Hyp-Gly-Gly,as a substrate.

The reaction was stopped by adding hydrochloric acid.

The released radio-labelled hyppuric acid was extracted with ethylacetate and counted by liquid scintillation spectrometry, according toconventional methods. The ACE-inhibitory activity of the compounds offormula I and of the compound R-1 was expressed as percentage ofinhibition in SHR lungs.

The percentage of NEP inhibition and of ACE inhibition, evaluated inkidneys and lungs of SHR respectively, of the compound 1 and of thecompound R-1 are reported int the following table 2.

Table 2

NEP-inhibitory and ACE-inhibitory activity, expressed as percentage ofinhibtion in SHR kidneys and lungs respectively, after i.v. injection(21 μmoles/kg) of the compound 1 and ofthe compound R-1.

ACE-inhibitory NEP-inhibitory activity activity (kidney) (lung) Compound% of inhibition % of inhibition 1 70 88 R-1 20 25

The data reported in table 2 clearly show that the compounds of formulaI, object of the present invention, are endowed with a dualACE/NEP-inhibitory activity significantly higher than that of thecompound R-1.

What is claimed is:
 1. A compound of formula

wherein R is a straight or branched C₁-C₆ alkyl group optionallysubstituted with one or more fluorine atoms, an aryl or arylalkyl groupwith from 1 to 6 carbon atoms in the alkyl moiety wherein the aryl is aphenyl, 1-naphthyl 2-napithyl group or a 5 or 6 membered aromaticheterocycle with 1 or 2 heteroatoms selected among nitrogen, oxygen andsulphur, optionally substituted with one or more substituents, the sameor different, selected among halogen atoms, hydroxy groups, alkylalkoxy, alkylthio, alkylsuphonyl or alkoxycarbonyl groups with from 1 to3 carbon atoms in the alkyl moiety, carboxy groups, aminocarbonylgroups, acylamino groups, aminosulphonyl groups, mono- ordi-alkylaminocarbonyl groups with from 1 to 3 carbon atoms in the alkylmoiety; R₁ and R₂, the same or different, represent a hydrogen atom or astraight or branched C₁-C₄ alkyl group; R₃ is a straight or branchedC₁-C₆ alkyl group or an arylalkyl group with from 1 to 6 carbon atoms inthe alkyl moiety wherein the aryl is a phenyl, 1-naphthyl, 2-naphthylgroup or a 5 or 6 membered aromatic heterocycle with one or twoheteroatoms selected among nitrogen, oxygen and sulphur, optionallysubstituted as indicated for R; R₄ is a 5 or 6 membered aromaticheterocyclic group with one or two heteroatoms selected among nitrogen,oxygen and sulphur, substituted with a 5 or 6 membered aromaticheterocyclic group with one or two heteroatoms selected among nitrogen,oxygen and sulphur or with a phenyl group, or it is a phenyl groupsubstituted with a 5 or 6 membered aromatic heterocyclic group with oneor two heteroatoms selected among nitrogen, oxygen and sulphur, whereinthe phenyl and the heterocyclic groups are optionally substituted withone or more substituents, the same or different, selected among halogenatoms, alkyl, alkoxy, alkylthio or alkoxycarbonyl groups with from 1 to3 carbon atoms in the alkyl moiety; X is a single bond or an —O—CONH— or—CONH— group; the carbon atoms marked with an asterisk are asymmetriccarbon atoms; and pharmaceutically acceptable salts thereof; providedthat R₄ is not an imidazolyl or indolyl group.
 2. A compound accordingto claim 1 wherein R₄ represents a phenyl group substituted in position4 with a heterocyclic group.
 3. A compound according to claim 2 whereinR₁ and R₂ represent a hydrogen atom and R₃ represents a straight orbranched C₁-C₄ alkyl group.
 4. A compound according to claim 1 in theform of a salt with an alkali metal selected among sodium, lithium andpotassium.
 5. A process for preparing a compound as recited in claim 1comprising (a) reacting a phosphorylated derivative of formula II

wherein R, R₁ and X have the meanings reported in claim 1, W representsa halogen atom and Y represents a protective group selected among aC₁-C₄ alkyl, a phenyl or a phenylalkyl with from 1 to 4 carbon atoms inthe alkyl moiety; with a dipeptide of formula III

wherein R₂, R₃, R₄ have the meanings reported in claim 1, and whereinthe carboxyl group is protected; for a time and under conditionseffective for the amino group of formula III to displace the halogenatom of formula II; (b) removing the protective groups; (c) isolatingthe compound of formula I.
 6. A compound of formula I according to claim1 selected betweenN-(N′-propylphosphonyl-L-leucyl-[4-(2-furyl)]-L-phenylalanine andN-(N′-propylphophonyl-L-valyl)-[4-(2-thiazolyl)]-L-phenylalanine.
 7. Acomposition comprising a compound according to claim 1 in combinationwith a pharmaceutically acceptable carrier.
 8. A method of inhibitingACE (angiotensin converting enzyme) and NEP (neutral endopeptidase)comprising administering a compound according to claim 1 to a patient inneed thereof for a time and under conditions effective to inhibit ACEand NEP.